A critical function of blood centres is the testing of blood to accurately determine the blood group type of the individual from whom the blood (or other product) was obtained. Knowledge of the blood group type is essential for a variety of therapies including blood transfusion, organ transplantation, and the treatment of haemolytic diseases of the newborn. In particular, an individual's blood group type must be determined prior to being given a blood transfusion. A mismatch of blood group types can have disastrous consequences potentially leading to the death of the transfused individual.
The ABO blood group system represents the most important of the antigens on human red blood cells (RBCs) for blood transfusion serology. Humans belong to one of four major groups: A, B, AB, and O. The RBCs of each group respectively carry the A antigen, the B antigen, both A and B antigens, or neither. Natural antibodies are present in the blood against the blood group antigen which is absent from the RBCs. Thus, individuals of group A have anti-B, those of group B have anti-A, those of group O have anti-A and anti-B, and those of group AB have neither. Before blood transfusion the blood must be cross-matched (either by testing the donor blood against the serum of the recipient or by electronically matching the blood against records) to ensure that RBCs of one group are not given to an individual possessing antibodies against them.
RBCs are tested against reagents containing known antibodies (known as forward grouping) and serum is tested against RBCs possessing known antigens (known as reverse grouping).
Monoclonal antibodies (MAbs) have been used as blood typing reagents since the 1980's. When compared with traditional polyclonal antisera, monoclonal reagents offer increased specificity, consistent reactivity, and, in most cases, increased potency.
Routine quality control of blood group systems (for example, gel cards) and reagents is essential in any blood bank laboratory. Reagents and blood grouping systems may suffer reductions in specificity or potency during shipping, storage, or as a result of contamination during storage and use.
Monoclonal reagents are required to identify all natural variations of ABO blood groups including subgroups of A and B. To ensure correct identification, monoclonal blood grouping reagents and blood grouping systems in blood bank laboratories are tested against RBC reagent controls. For this purpose, RBCs with a weak antigen expression are preferred as the control reagent. This is because such RBCs can provide a better indication of an antiserum's potency for the identification of weak phenotypes.
There exist in nature various forms of weak or poorly expressing ABO subgroups. The A/B antigen concentrations within each of the cell phenotypes are variable and generally unknown unless extensive analysis is performed.
Using weak phenotyping RBCs as control reagents is difficult in practice, due to the very low frequency of subgroup phenotype individuals. For example, the Ax phenotype is estimated as 0.003% of group A and other subgroups have even lower frequency. Artificial weak phenotype RBCs may therefore be useful for this purpose.
Group O RBCs transformed into artificial group A RBCs or group B RBCs or group AB RBCs appear to resemble weak phenotypes serologically. Expression of these antigens can be controlled by changing the insertion conditions, such as the concentration of inserted antigen, and/or the ratio of RBC to antigen for insertion or amount of synthetic antigen added etc. The inserted antigens can be stable in the RBC membrane in certain conditions for at least six weeks, and possibly longer.
Currently, serological sensitivity of monoclonal antibodies (antisera) used for the detection of cells that poorly express carbohydrate antigens can be determined in one of several ways:    1. Testing against natural weak subgroups. This involves finding a rare subgroup, preparing cells of this subgroup ready for use, and then using them as controls.    2. Testing against normal cells. This involves testing common cells and does not give any indication of sensitivity.    3. Diluting antisera to determine potency. This involves diluting antibodies and testing against normal antigens. This is the most common practice in the absence of true controls.
Natural cells, due to their frequency, are very difficult to obtain and maintain supply. In addition, they vary between individuals. Constant supply would be difficult, if not impractical. Further, different populations have different frequencies of weak subgroups.
Normal cells express high levels of antigen, for example in the region of >500,000 copies per red cell. When testing these cells, the reagents are typically diluted to show that at low dilution they can still react with RBCs and give a serologically positive result. This dilution sensitivity method is time consuming. The results are then extrapolated to determine the detection level of antigen at normal dilution. This flawed methodology is unfortunately the practice in most places. Detection of reagent deterioration would only be possible if regular time consuming dilution studies were undertaken or weak subgroups were tested.
Additional problems can occur with the dilution of antisera. Monoclonal reagents are often biclonal and formulated to give specific performance characteristics. It is well known that some clones are better than others at detecting ABO subgroups. As a consequence, reagents are often formulated as blends. Dilution of such reagents negates their intrinsic performance features and thus will not reflect the true performance of the reagents Furthermore, many monoclonal reagents now come formulated for and pre-loaded into test card systems (i.e. gel cards) and thus cannot be tested by dilution methods.
Many laboratories do not presently routinely carry out sensitivity controlling of their ABO blood typing reagents. Instead they rely upon the manufacturer and the historical performance of the reagents. Alternatively, laboratories may only batch test on a weekly or even monthly basis in the manner described for 3 above. Furthermore, many rely on the literature outcomes of accidental transfusion of a weak subgroup to an incompatible recipient, which indicates that these events are usually non-fatal. Previously, a cross-match (the testing of the donor's blood against the recipient's serum) would detect an incompatibility between a weak subgroup mistyped and for transfusion to an incompatible recipient. However, these days cross-matching is not performed in many centres and instead correct blood typing of both the donor and recipient is relied upon. It is therefore now more important that blood is accurately typed. The problem of not carrying out any testing is that the blood typing reagents may have deteriorated and a clinically significant subgroup may be incorrectly blood typed in the absence of cross-matching. Such blood may cause a mild to severe transfusion reaction.
There is therefore a clear need for sensitivity control reagents which have a known predetermined amount of antigen expression and are therefore capable of being used to calibrate testing reagents or testing systems to give accurate and standardised determinations of blood group types.
It is an object of this invention to provide a sensitivity control reagent for blood group determinations, or to at least provide a useful alternative.